CRISPRi/a functional screen with PARP7 inhibitor in NCI-H1373 cells

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. PARP7 expression is increased by aromatic hydrocarbons and cellular stress, and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 is a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores Type I IFN signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. RBN-2397 is a potent and selective inhibitor of PARP7. To further explore how RBN-2397 inhibits NCI-H1373 cell proliferation, we performed unbiased genetic screens using whole-genome CRISPRi and CRISPRa libraries (le Sage et al., 2017; Jost and Weissman, 2018). Comparison of CRISPRi and CRISPRa phenotype scores highlights genes with opposing functionality upon RBN-2397 treatment. CRISPR-Cas9 dual screening with RBN-2397 (2.5 nM) in NCI-H1373 cells was performed at Horizon Discovery (Cambridge, UK). Briefly, cells were transduced with either whole-genome CRISPR activation (CRISPRa) or interference (CRISPRi) libraries, then selected and maintained in culture to allow CRISPR-driven gene perturbations to occur (le Sage et al., 2017). Genomic DNA was extracted using the QIAamp DNA Blood Maxi kit (Qiagen #51194). Samples were prepared and purified for amplicon sequencing using an Illumina NextSeq next generation sequencing platform. Analysis of NGS data sets e.g. sgRNA abundance was achieved using Horizon’s data processing scripts, based on published analysis tool MAGeCK v0.5.04,5. Dual screen analysis was achieved by normalizing average log-fold changes in guide abundance by the difference in population doubling for each screen track: Phenotype score = LFC (DMSO vs RBN)/ΔPD (DMSO vs RBN). Vehicle (DMSO) treated cells were used as control.